One of the principal mechanisms by which cellular regulation is effected is through the transduction of extracellular signals across the membrane that in turn modulate biochemical pathways within the cell. Protein phosphorylation represents one course by which intracellular signals are propagated from molecule to molecule resulting finally in a cellular response. These signal transduction cascades are highly regulated and often overlapping as evidenced by the existence of many protein kinases as well as phosphatases. It is currently believed that a number of disease states and/or disorders are a result of either aberrant activation or functional mutations in the molecular components of kinase cascades. Consequently, considerable attention has been devoted to the characterization of these proteins.
Nearly all cell surface receptors use one or more of the MAP kinase cascades during signal transduction. One subgroup of the MAP kinases is the MAPK/ERK kinases or MEKs. There are five members of the MEK family, two of which, MEK1 and MEK2, lie directly downstream of the Raf oncogene pathway. However, several other upstream components have been shown to activate MEKs including kinases in the c-Mos, and MEKK pathways (Robinson and Cobb, Curr. Opin. Cell Biol., 1997, 9, 180-186). MEKs catalyze both serine/threonine and tyrosine phosphorylation of their target molecules and do so in an ordered fashion (Seger et al., J. Biol. Chem., 1992, 267, 14373-14381).
MEK2 (also known as MAPKK2, mpk2, MAPK/ERK kinase 2, prkmk2, and erk activator kinase 2), is a member of the MEK family of dual-specificity kinases and represents a convergent target for the regulation of a diverse set of cellular processes including proliferation, differentiation and development. MEK2 was first identified as an ERK activator in growth factor treated cells and was later cloned and characterized as a mitogen-activated protein kinase of ERK (Zheng and Guan, J. Biol. Chem., 1993, 268, 11435-11439). MEK2 mRNA is expressed in high levels in embryonic tissues, including the liver and neural tissue. However, it has low expression levels in adult tissues, implying that MEK2 may be the primary activator during development while other MEKs mediate the proliferative or mitogenic responses in the adult (Brott et al., Cell Growth Differ., 1993, 4, 921-929). Downstream targets of MEK2 identified to date are ERK1 and ERK2. Subsequent to phosphorylation, ERKs activate nuclear, membrane, cytosolic and cytoskeletal targets that in turn mediate multiple signaling cascades (Seger and Krebs, The FASEB Journal, 1995, 9, 726-735). Therefore, manifestations of altered MEK2 regulation can appear in many downstream events the most widely investigated being the development of cancer.
MEK2 has been shown to be overexpressed in particulate and cytosolic fractions prepared from tumor specimens in human hepatocellular carcinoma (Schmidt et al., Biochem Biophys Res Commun, 1997, 236, 54-58). In studies of drug sensitive human breast carcinoma cells there was a three fold increase in MEK2 activation upon glucose deprivation suggesting that glucose deprivation induces a MAPK pathway that may be responsible for the maintenance of carcinoma (Gupta et al., Mol. Cell Diochem., 1997, 170, 23-30). Other studies have demonstrated that oncogenes capable of transforming mammary gland epithelium require specific signal transduction pathways and that mammary tumors initiated by neu, v-Ha-ras, and c-myc have high levels of MEKs. Furthermore, the anchorage independent growth of these tumors was inhibited by the MEK specific inhibitor, PD 098059 (Amundadottir and Leder, Oncogene, 1998, 16, 737-746).
To date, strategies aimed at inhibiting MEK2 function have involved the use of anti-MAPKK antibodies, the chemical inhibitor PD 098059 and dominant-negative forms of MEK2.
The most widely used inhibitor of MEK2 is the chemical moiety, PD 098059. This compound has been used extensively to demonstrate the involvement of MEKs in signaling cascades. It was shown to act as a noncompetitive inhibitor of MEKs by blocking activation by Raf and MEK kinases (Alessi et al., J Biol Chem, 1995, 270, 27489-27494). However, it has recently been proposed that this highly selective inhibitor of MEK2 competes with apoptotic signals thereby producing cells that avoid apoptosis and contribute to pathologic conditions (Mohr et al., Proc. Natl. Acad. Sci. U S A, 1998, 95, 5045-5050).
In studies of xenopus oocyte maturation, microinjection of anti-MAPKK neutralizing antibodies prevented the mos-induced metaphase arrest of the cell cycle, implicating MEK in cell cycle control (Kosako et al., J Biol Chem, 1994, 269, 28354-28358).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of MEK2. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting MEK2 function.
Antisense oligonucleotides, therefore, provide a promising new pharmaceutical tool for the effective modification of the expression of specific genes.